Cloning And Characterization Of Novel Virulence Genes From Xanthomonas Axonopodis Pv.Citri

A genome-wide isolation of novel virulence genes was carried out by Tn5-based random mutagenesis in Xanthomonas axonopodis pv. citri（Xac）.46 mutants were identified to be altered in virulence from approximately20000 mutants. Plasmid rescue experiment revealed that these mutants corresponded to 22 ORFs. Complementation analysis was subsequently performed on 7 novel virulence genes. Three mutants Mxac154-24,Mxac110-56 and Mxac122-63 were fully impaired in pathogenicity in citrus and the ability to induce hypersensitive response in nonhost plants. Mutant Mxac154-24 had an insertion in carbamoyl phosphate synthase large subunit（carB）, while the other two mutants with insertion in carbamoyl phosphate synthase small subunit（carA）. Deletion mutant ΔcarA, ΔcarB and Δcar AcarB were constructed for further phenotypic analysis, including virulence,hypersensitive response induction, motility, and extracellular enzyme activity.RT-PCR results indicated that carA XAC 29₀1863 car B greA and rpf E were located within one transcription operon.In our previous work, an extracellular protease mutant ΔecpA of X.oryzae pv. oryzicola（Xoc） showed a complete loss of protease activity. The homologous gene in X. oryzae pv. oryzae had an extra 5 nucleotides in C-terminals, resulting in a shift reading in relative with Xoc Ecp A. A mutant deletion in Xac ecpA retained partial extracellular protease activity, indicating that Xac may have more than one extracellular protease coding genes.